A placenta de lhama tem sido descrita como epitélio-corial, mas os estudos existentes não aprofundam nos aspectos microscópicos. Para detalhar as características microestruturais foram feitas observações ao microscópio de luz, eletrônico de varredura e de transmissão. Foram coletados 09 úteros grávidos em associação com as membranas fetais, entre 28 a 36 semanas de gestação. Parte do material foi fixado com solução de paraformaldeído 4% e emblocado em paraplast e historesina. Seções de 5µm foram submetidas a colorações de hematoxilina-eosina, Tricrômico de Masson, reações histoquímicas de PAS, Perl?s e fosfatase ácida, e imunohistoquímica para a detecção de uteroferrina. Fragmentos foram fixados com glutaraldeído 2,5%, para microscopia eletrônica de varredura e transmissão. Os resultados observados permitem classificar a placenta da lhama como corioalantóidea, difusa, pregueada, epitéliocorial e o feto esta recoberto pela membrana epidermal. O trofoblasto apresentou células de morfologia variada, desde cúbicas, arredondas até triangulares, com citoplasma contendo grânulos PAS+. Células binucleadas com citoplasma aumentado e núcleos arredondados, bem como células trofoblásticas gigantes com múltiplos núcleos, também foram observadas. As aréolas estavam preenchidas de material PAS positivo Observaram-se grande quantidade de vasos sangüíneos na interface materno-fetal, entre as células do epitélio uterino e ao arredor das projeções coriônicas, as quais eram ramificadas. O mesênquima com fibras colágenas em grande quantidade auxiliam no suporte das projeções coriônicas. Observou-se positividade as reações histoquímicas de PAS, Perl?s e fosfatase ácida, e imunohistoquímica de uteroferrina na interface materno fetal, mas com maior notoriedade no epitélio e lume das glândulas uterinas. Células trofoblásticas gigantes com 4 ou mais núcleos estavam nas projeções coriônicas, mostrando escassa ou nula reatividade histoquímica ao PAS, indicando funcionalidades diferentes das células trofoblásticas mono ou binucleadas. O alantóide estava conformado por uma camada única de células de diferentes alturas, sendo que na superfície destas observaram-se estruturas circulares e poliédricas. A membrana epidermal possui um epitélio estratificado plano de até sete camadas de células mono, bi ou trinucleadas. A alta vascularização das faces materna e fetal, a qual indica uma ótima troca de sustâncias entre ambas faces, e a alta atividade metabólica demonstrada nas glândulas uterinas revelam uma adaptação da gestação desta espécie habitante das altas altitudes da serra do Peru. / The placenta of the llama has been described like epitheliochorial, but existent researches don\'t study in depth microscopic aspects. In order to detail their ultrastructurals characteristics observations were made by light microscopy and scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Samples of nine uteruses between 28 to 36 weeks of pregnancy were collected in association with fetal membranes. A part was fixed with of 4% paraformaldehyde solution and embedded either in paraffin or in glycol methacrylate resin. Sections of 5µm in thickness were stained with haematoxylin and eosin, Masson\'s trichrome staining and PAS (Perl`s and acid fosfatase, and inmunohistochemestry for uteroferrina detection. Another part was fixed with 2,5 % glutaraldehyde and post-fixed in 1% osmium tetroxide and processed for SEM. The trophoblast presented cells of morphology varied, since cubical, rounds off until triangular, with cytoplasm with granules PAS+. Binucleates cells with increased cytoplasm and spherical nucleus, as well as giant trophoblastics cells with multiple nucleus, had been also observed. Aréolas was filled of positive material PAS. Great amount of blood vessels are in the maternofetal interface, between the cells of uterine epithelium and around of the chorionic projections. Collagen fibers are observed in the mesenchymae and inside the chorionic projections. PAS positive reaction was observed in the materno-fetal interface, mane manifest in epithelium and lumen of uterine glands. Giant size trofoblastic cels of 4-7 or more nucleus were found in chorionich proyections, showing scarse or nule histochemistry reactivity, indicating different functionalities of different kind of mono or bi nucleated cells. The allantois is structured by an unique layer of different sizes cells, in their surface are circular and polyhedral structures, representing a morphological consideration from this type of function allantois cells. The epidermal membrane is formed by 7 or more layers of cells with one, two or three nucleus. The superficial layer present more smooth cells. The high vascularization of the maternal and fetal faces indicates an optimal interchange of substances between both. The collagen inside the chorionics projections serves as a support skeleton), and the high metabolic demonstrate activity in uterine glands, develope a pregnacy adaptation of these specie who lives in highs of Perú.

Neste estudo caracterizamos a expressão de Mif nas células trofoblásticas (CT), placentárias e decidua (D) na gestação em camundongos.A imunolocalização foi realizada nos dias de gestação(dg) 7,5, 10,5, 13,5 e 17,5.Com cones ectoplacentários e placentas fetais (PL) realizou-se ensaios de Western blotting e qRT-PCR nos mesmos dg.D foram submetidas a qPCR.Aos 7,5 dg as CT gigantes e D apresentaram imunomarcação.Dos 10,5 ao 17,5 dg o Mif concentrou-se nas CTG e no espongiotrofoblasto.Na D, a imunomarcação foi menor que aos 7,5 dg.A expressão protéica na PL aumentou do 7,5 dg para o 10,5 dg (p=0,005) e para o 13,5 dg (p=0.03).A maior expressão gênica foi em 10,5 dg e diferente em 13,5 dg (p=0,048) e 17,5 dg (p=0,009).Na D, a maior expressão gênica foi em 7,5 dg e diferente dos 10,5 (0,012) e 13,5 (0,032) dg.O aumento da expressão de Mif na PL coincide com a organização em quatro camadas e com o início da circulação fetal.Esta distribuição temporal e tecido-específica sugere o MIF como modulador no início da placentação ou na sua adaptação ao ambiente uterino / The goal of this study was to characterize Mif expression by trophoblast (TC),fetal placenta (FP) and decidua (D) during mouse pregnancy.Mif was immunolocalized at TC and D on gestation days (gd) 7.5, 10.5, 13.5 and 17.5.Ectoplacental cones (EC) and FP were used for Western blotting and qPCR.D were also used for qRT-PCR.On gd 7.5,DC and TC giant (TGC) showed strong reactivity.On gds 10.517.5,were concentrated in TGC and spongiotrophoblast cells. D reactivity was weaker than 7.5 gd.Protein expression at FP increased from gd 7.5 to 10.5 (p=0.005) and to 13.5 (p=0.03).Higher mRNA expression was found on gd 10.5 and was different from gds 13.5 (p=0.048) and 17.5 (p=0.009).At D on gd 7.5 was greater than those on gds 10.5 (0,012) and 13.5 (0,032).The up-regulation of Mif coincides with the stage that placenta assumes its four-layered organization and the fetal blood circulation begins,This temporal tissue-specific distribution and expression data suggests that Mif may play a modulator role in the onset of placentation or in it adaptation to the uterine environment

Somatic cell nuclear transfer (SCNT), or cloning, is a form of artificial reproductive technology that can be used to improve economic traits of domestic animals. However, extreme inefficiency of producing viable offspring via this method is a major limitation. An aggressive immune response at the maternal-fetal interface is an important reason for SCNT pregnancy loss. The goal of this project was to investigate the molecular mechanisms of immune-mediated miscarriage in cloned cattle pregnancies. Many publications hint that immune-mediated miscarriage is associated with abnormal MHC-I expression in the placenta. The regulation of bovine MHC-I genes was systematically studied to identify the cause of abnormal MHC-I expression during immune-mediated miscarriage. We also produced cloned pregnancies to study immune- mediated pregnancy loss. MHC-I and cytokines involved in proinflammatory responses were highly expressed in the placental trophoblast cells of cloned fetuses and in the uterine endometrium of recipients carrying MHC-I incompatible fetuses, respectively, suggesting that MHC-I compatibility between fetus and surrogate mother is important for the success of animal cloning. The results from this research not only reveal the cause of high pregnancy loss in cloned animals but also provide molecular clues to prevent immune-mediated miscarriage in cattle and potentially in human clinics.

Human papillomavirus (HPV) is a double-stranded DNA virus, typically infecting mucosal or cutaneous epithelial keratinocytes. Today, more than 118 different HPV types have been formally described. Sexual transmission of mucosal HPVs is very common and generally asymptomatic, but HPV infection can be associated with benign lesions such as condylomata acuminata or, in rare cases, with malignant lesions such as cervical cancer. Two prophylactic vaccines are currently available in Europe, protecting against HPV-16 and HPV-18 (Cervarix) or against HPV-6, HPV-11, HPV-16 and HPV-18 (Gardasil). In order to assess the impact of the vaccination program, it is mandatory to obtain geographically widespread date on the baseline HPV prevalence and type distribution in cervical samples from women, presenting or not, normal or abnormal cytologic or histologic results. We undertook an epidemiological study in the Capital Region of Brussels to determine the HPV prevalence and type-distribution in 1526 cervical samples of women presenting a cytology within normal limits (WINL), atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intra-epithelial lesions (LSIL), high-grade squamous intra-epithelial lesions (HSIL) or invasive cervical cancer (ICC). The HPV prevalence was 10.8% (95%CI: 8.8-12.8) for NILM, 34.5% (95%CI: 28.3-40.8) for ASC-US, 54.0% (95%CI: 47.4-60.6) for LSIL and 100% for HSIL and ICC. With an HPV-16 and HPV-18 prevalence of 63.3% (95%CI: 44.1-67.7) and 73.5% (95%CI: 63.0-84.0) in mono-infected HSIL and ICC, respectively, HPV 16/18 L1 VLP vaccines would be expected to significantly reduce the management and treatment of women suffering from HSIL and ICC in the Capital Region of Brussels. We also detected HPV-30, HPV-53, HPV-66 and HPV-68 in mono-infected HSIL and ICC samples, possibly providing arguments for the reconsideration of the carcinogenicity of these types. Vertical transmission of HPV was also previously reported, but in most cases one could not exclude a placental contamination by HPV positive cells from an infected birth canal. In order to confirm that the placenta can be infected with HPV, we analysed residual cells from 35 transabdominally obtained placental samples from pregnant women undergoing chorionic villous sampling for screening of suspected foetal abnormalities and found that two samples were positive for HPV-16 and HPV-62, respectively. The clinical importance of these results remains to be elucidated, but the previously observed association between placental HPV infection and pregnancy loss might gain further in importance. HPV gene regulation in placental trophoblastic cells has not been studied so far. We studied the HPV-16 early gene expression regulation in transiently transfected monolayer cultured trophoblastic cells with an HPV-16 long control region (LCR) driven reporter plasmid. We observed important differences in constitutive HPV-16 LCR activities between trophoblastic cell lines and could identify progesterone as an important inducer of HPV-16 early gene expression. Steroid hormones are induced during pregnancy and could therefore lead to an enhanced expression of the E5, E6 and E7 proteins upon placental HPV infection. Since these proteins were previously shown to affect trophoblast adhesion, survival, migration and invasion, their enhanced expression might eventually contribute to pregnancy loss. We furthermore found that the transcription of episomally maintained HPV-16 is not regulated by E2 or E1, but by E5, E6 and/or E7.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 171-195). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 171-195). Also available on the Internet.

Thesis (M. Sc.)--York University, 2001. / Typescript. Includes bibliographical references (leaves 69-89). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ67745.

MicroRNAs are small regulatory RNAs that bind to the seeding regions within the 3’-untranslated region (3’-UTR) of their target transcripts to modulate transcript stability and/or inhibit protein translation. MicroRNA Let-7a belonging to the Lethal-7 (Let-7) family is down-regulated at the blastocyst stage, suggesting its suppression is crucial for embryo implantation. Yet, the underlying mechanism on how Let-7a modulates blastocyst implantation remains largely unknown. In silico analysis identified attachment related integrin beta-3, outgrowth related vav3, and the microRNA processing dicer, as Let-7a targets. Therefore, it is hypothesized that down-regulation of Let-7a promotes embryo implantation by stimulating these target proteins. Let-7a is down-regulated during blastulation and at 3-hour post-estradiol activation of the dormant blastocysts. Force-expression of Let-7a in mouse blastocysts suppressed blastocyst attachment, outgrowth on fibronectin-coated plates and compromised pregnancy in vivo. Dual luciferase assay using the 3’-UTR reporter constructs of the integrin beta-3, vav3, and dicer confirmed the interaction between Let-7a and the three targets. Force-expressing or inhibiting Let-7a expression in mouse blastocysts by electroporating the Let-7a precursor or inhibitor respectively illustrated post-transcriptional regulation of Let-7a on integrin beta-3 and vav3, and transcriptional regulation on dicer. Dormant blastocysts retrieved from the delayed implanting mice expressed high Let-7a levels, which was suppressed in the first 12-hours of estradiol activation. Concomitantly, dormant blastocysts expressed low levels of integrin beta-3, vav3, and dicer, yet, their protein expression was up-regulated from 3 hours-post estradiol activation. Furthermore, addition of integrin beta-3 antibody suppressed attachment of trophoblast spheroids (blastocyst surrogate) onto endometrial epithelial cells in a co-culture model and the outgrowth of the spheroids on fibronectin-coated plates. Knockdown of Vav3 with siRNA decreased blastulation, hatching, and outgrowth rates of the embryos in vitro. Although the loss of vav3 activities did not affect embryo implantation, it suppressed trophoblast migration on fibronectin-coated plates and invasion into collagen matrix. In contrast, force-expression of vav3 enhanced blastocyst outgrowth, and promoted cell proliferation. Blocking integrin beta-3 activities in the vav3 knock-down embryos further suppressed blastocyst outgrowth, suggesting the intertwining effect of the integrins and vav3. Dicer knock-down in mouse blastocysts decreased mature Let-7a expression and suppressed blastulation and hatching in vitro and implantation in vivo. Dicer knock-down in estradiol activated mouse blastocysts decreased the epidermal growth factor receptor expression and lowered the affinity of the embryos to EGF, and suppressed the implantation potential to a level similar to that of dormant blastocysts. Taken together, the suppression of Let-7a by estradiol up-regulates integrin beta-3, vav3, and dicer. The increased Itgb3 expression promotes blastocyst attachment and intertwined with the up-regulated vav3 expression to enhance blastocyst outgrowth. The increased vav3 expression further stimulates cell proliferation and confers blastocyst invasion into the collagen matrix. Dicer, by altering microRNA processing, facilitates blastulation and thereby embryo implantation. Thus, the loss of Let-7a biological activities during blastulation is crucial to enhance blastulation and stimulate trophoblast activities for successful embryo implantation. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Physiology / Master / Master of Philosophy

Title from PDF of title page (University of Missouri--Columbia, viewed on February 23, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Jonathan A. Green. Vita Includes bibliographical references.